Resveratrol modulates gene expression associated with apoptosis, proliferation and cell cycle in cells with mutated human c-Ha-Ras, but does not alter c-Ha-Ras mRNA or protein expression

An accumulating body of evidence suggests that resveratrol can inhibit carcinogenesis through antiproliferative and apoptotic effects. One proposed mechanism for this is the modulation of genes, for example, Ras and p53, frequently associated with human cancer. To test the effect of resveratrol on gene expression, we used the WR-21 cell line because it contains a mutated human c-Ha-ras gene. Cells at >= 70% confluency were incubated with media alone or with increasing concentrations of trans-resveratrol (0.1-1000 mu M) for 24 h. Resveratrol (30-100 mu M) decreased cellular proliferation by 80% (bromodeoxyuridine incorporation) and increased apoptosis by 60% (TUNEL). Cells were then treated with media alone or with 50-mu M resveratrol for 24 h. RNA was isolated for nylon-based macroarray analyses and protein for immunoblotting. Resveratrol increased (+) and decreased (-) gene expression associated with apoptosis (Birc5+, Cash+, Mcl-1+, Mdm2+, Rpa-like+), cellular proliferation (Ctsd+, Mdm2+, Egr1+, ODC+) and cell cycle (cyclin D+, cyclin g+, Gadd45a-, Mad21-, Mdm2+). Resveratrol consistently increased by >= 6-fold Mdm2 expression and other downstream p53 effectors, but not p53 itself at 24 h. Subsequent cell cycle analysis indicated a significant accumulation of cells in G2/M, and a decrease in G1/G0 suggesting a G2/M blockade. Further RTPCR and Western blot analyses indicated no differential changes in Ras mRNA expression or p21(ras) protein levels, respectively. These results suggest that resveratrol potently inhibits cellular proliferation, increases apoptosis, alters cell cycle dynamics and modulates associated gene expression. Furthermore, these effects appear mediated, in part, by p53 without direct modulation of mutant c-Ha-ras expression. (c) 2005 Elsevier Inc. All fights reserved.